Establishment of a monoclonal antibody for human LXRα: Detection of LXRα protein expression in human macrophages

Liver X activated receptor alpha (LXRalpha) forms a functional dimeric nuclear receptor with RXR that regulates the metabolism of several important lipids, including cholesterol and bile acids. As compared with RXR, the LXRalpha protein level in the cell is low and the LXRalpha protein itself is very hard to detect. We have previously reported that the mRNA for LXRalpha is highly expressed in human cultured macrophages. In order to confirm the presence of the LXRalpha protein in the human macrophage, we have established a monoclonal antibody against LXRalpha, K-8607. The binding of mAb K-8607 to the human LXRalpha protein was confirmed by a wide variety of different techniques, including immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). By immunoblotting with this antibody, the presence of native LXR protein in primary cultured human macrophage was demonstrated, as was its absence in human monocytes. This monoclonal anti-LXRalpha antibody should prove to be a useful tool in the analysis of the human LXRalpha protein.     

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.     

Lipopolysaccharide induces physical proximity between CD14 and toll-like receptor 4 (TLR4) prior to nuclear translocation of NF-kappa B

CD14, a GPI-linked protein, plays a pivotal role in LPS-mediated signaling by potentiating leukocyte adherence, activation, and cytokine production. Recent studies have identified the Toll-like receptor 4 (TLR4) as a membrane cofactor in LPS-mediated transmembrane signaling in cytokine induction, although the mechanism responsible for this cooperation is unknown. Using fluorescence resonance energy transfer (RET) techniques, we demonstrate that LPS triggers a physical association between CD14 and TLR4. Because LPS stimulation upregulates CD14 and TLR4 expression, it was necessary to control for the possibility that these newly expressed molecules were associated with one another independent of LPS stimulation. Although the calcium ionophore A23187 increased the expression of CD14 and TLR4, they did not exhibit energy transfer. However, following A23187 treatment, LPS promoted physical proximity between CD14 and TLR4. Therefore, we suggest that a close interaction between CD14 and TLR4 participates in LPS signaling, leading to nuclear translocation of NF-kappaB.     

Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression

Monocytes/macrophages exposed to LPS show reduced responses to second stimulation with LPS, which is termed LPS tolerance. In this study, we investigated molecular mechanism of LPS tolerance in macrophages. Mouse peritoneal macrophages pre-exposed to LPS exhibited reduced production of inflammatory cytokines in a time- and dose-dependent manner. Activation of neither IL-1 receptor-associated kinase nor NF-kappaB was observed in macrophages that became tolerant by LPS pretreatment, indicating that the proximal event in Toll-like receptor 4 (TLR4)-MyD88-dependent signaling is affected in tolerant macrophages. Although TLR4 mRNA expression significantly decreased within a few hours of LPS pretreatment and returned to the original level at 24 h, the surface TLR4 expression began to decrease within 1 h, with a gradual decrease after that, and remained suppressed over 24 h. A decrease in inflammatory cytokine production in tolerant macrophages well correlates with down-regulation of the surface TLR4 expression, which may explain one of the mechanisms for LPS tolerance.     

Neospora caninum:Tachyzoites Express a Potent Type-I Nucleoside Triphosphate Hydrolase,but Lack Nucleoside Diphosphate Hydrolase Activity

Asai, T., Howe, D. K., Nakajima, K., Nozaki, T., Takeuchi, T., and Sibley, L. D.Neospora caninum: Tachyzoites Express Type-I Nucleoside Triphosphate Hydrolase1. But Lack Nucleoside Diphosphate Hydrolase Activity.Experimental Parasitology90,277–285. We have identified type I nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) activity, previously thought to be restricted to the virulent strains ofToxoplasma gondii, in the cell extracts ofNeospora caninumtachyzoites. Sequence analysis of a complete cDNA from Nc-1 strain indicated thatN. caninumNTPases shared approximately 69% identity to the NTPases ofT. gondiiand are most similar to the NTPase-I isozyme. Southern blot analysis of genomic DNA and sequence analysis of two independentNTPclones from the Nc-1 strain revealed the presence of multiple genes, at least two of which are transcribed. Substrate specificity andK_mvalues for MgATP²⁻and MgADP⁻hydrolysis for recombinant or partially purified native NcNTPase were the same as those for the type I isozyme (NTPase-I). Significantly, no type II enzyme (NTPase-II) activity for NDP hydrolysis was detected in cell extracts ofN. caninum, although it is universally present in allT. gondiistrains that have been tested. This intriguing difference between these two closely related apicomplexan parasites may provide insight into the function of the NTPases during intracellular parasitism.     

Translational selection and molecular evolution

An interplay among experimental studies of protein synthesis, evolutionary theory, and comparisons of DNA sequence data has shed light on the roles of natural selection and genetic drift in ‘silent’ DNA evolution.     

Low molecular weight chitosan-g-l-phenylalanine: Preparation,characterization, and complex formation with DNA

The grafting of l-phenylalanine onto low molecular weight chitosan is accomplished by using carbodiimide as a coupling agent. As increase in the amount of phenylalanine in feed, the grafting chain length increases, while a number of grafting chains hardly change. The obtained product, LMWCts-g-Phe, performs sphere with an average size of ～80 nm when the % grafting is less than 123. The complexes of the LMWCts-g-Phe and DNA (LMWCts-g-Phe/DNA) prepared by a complex coacervation method possess various shapes with an average size of ～50–150 nm and a negatively charged surface. The LMWCts-g-Phe and its complex show very reduced toxicity to fibroblast cells. The release of DNA from the complex is very fast in high pH media (tris buffer, pH 8.0 and carbonate buffer, pH 9.5), and relatively slow or more sustainable in neutral and low pH ones (PBS, pH 7.4 and citric acid/trisodium citrate buffer, pH 3.0). The results suggest that the LMWCts-g-Phe be an alternative promising carrier for negatively charged active molecules.     

Assessment of the Annual Additional Effective Doses amongst Minamisoma Children during the Second Year after the Fukushima Daiichi Nuclear Power Plant Disaster

An assessment of the external and internal radiation exposure levels, which includes calculation of effective doses from chronic radiation exposure and assessment of long-term radiation-related health risks, has become mandatory for residents living near the nuclear power plant in Fukushima, Japan. Data for all primary and secondary children in Minamisoma who participated in both external and internal screening programs were employed to assess the annual additional effective dose acquired due to the Fukushima Daiichi nuclear power plant disaster. In total, 881 children took part in both internal and external radiation exposure screening programs between 1^st April 2012 to 31^st March 2013. The level of additional effective doses ranged from 0.025 to 3.49 mSv/year with the median of 0.70 mSv/year. While 99.7% of the children (n = 878) were not detected with internal contamination, 90.3% of the additional effective doses was the result of external radiation exposure. This finding is relatively consistent with the doses estimated by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR). The present study showed that the level of annual additional effective doses among children in Minamisoma has been low, even after the inter-individual differences were taken into account. The dose from internal radiation exposure was negligible presumably due to the success of contaminated food control.     

Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3′-Phosphoadenosine 5′-Phosphosulfate and ATP

Entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3′-phosphoadenosine 5′-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba, PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids.